Correlaton Of DNMT1 And DAPK In Cervical Carcinoma And Inhibition Of DNMT1 In Vitro

Cervical carcinoma(CC) is the second most common carcinoma and a leading cause of cancer-related death in women worldwide.Recently,the patients with cervical carcinoma are younger than ever.Genital HPV(human papillomavirus) infection has been identified as an important element in cervical carcinogenesis.Other factors are also involved in cervical carcinogenesis,since the majority of patients infected with HPV do not develop invasive cervical carcinoma.Molecular basis of the etiology of cervical carcinoma is still poorly understood.In recent years,surgery is the major treatment to cervical carcinoma.Excision can not cut out all the tumor cells,though adding to radiation and chemotherapy because of tumors' invasive characteristic,so the relapse rate and mortality are still high.Finding the best therapy for patients with cervical carcinoma is a dream for gynecological oncologists.To resolve it,the key point is to uncover the molecular mechanism of cervical carcinogenesis.The modern oncological viewpoint believes that the occurrence of tumor is a multi-factor,multi-step and multi-stage complex process,not only related with the hereditary changes,but also with the aberrance of epigenetics.The epigenetics refers not to change the DNA sequence,but through reversible modification of nucleotide or chromosomes to regulate gene expression,and the modification is heritable,including DNA methylation,genomic imprinting and histone modification.The most widely studied epigenetic modification in humans is DNA methylation,which plays an important role in the occurrence and the development of the tumor.The DNA methylation is a process,which through the DNA methyltransrase catalyzing,taking S-the adenosine-L- methionine(S-adenosyl-L-methionine,SAM) as the methyl donor,shifting the methyl to the cytosine 5 carbon atoms,produces 5- methyl cytosine(5-methylcytosine,5mC).In the normal state,the promoter region of the CpG island is non-methylation,and gene expression can be normal,but when it occurs methylation,there is an effect in transcriptional regulation of gene,which often leads to silence of gene expression and important genes such as tumor suppressor genes,DNA repair genes,resulting in abnormal regulation of cell growth and differentiation.The aberrance of DNA methylation in the promoter region of the CpG Island plays an important role in the occurrence and progression of tumor.At present,five kinds DNMTs,namely DNMT1,DNMT2,DNMT3a,DNMT3b and DNMT3L have been found in eukaryotes.Among them,DNMT1 is the most important one.The methylation modification undering DMNT1 play an important role in regulating cell growth,such as participating in the DNA replication repair,regulating embryonic development,X chromosome gene of the feminine deactivation,genomic imprinting and inhibiting the expression of exogenous sequences,and so on.In general,DNMT1 is lowly expressed in normal tissue,but highly expressed in a variety of solid tumors and hematopoietic system tumors,accompanied by the increased enzyme activity.Therefore, DNMT1 is closely related to the tumor,and it is expected to become an important target for tumor therapy.Inhibition of DNMT1 activity has become a reversal of aberrant methylation from the perspective of an important means to treat cancer.As an apoptosis gene,DAPK can mediate cell apoptosis induced by various stimulations.DAPK functions as a tumor suppressor gene by promoting cell apoptosis. Recently,the aberrant promoter hypermethylation of DAPK gene in tumors attracted more attention.Hypermethylation of DAPK gene involving in many primary tumors indicates that the hypermethylation of CpG ilands in the promoter of DAPK gene plays an essential role in the development of tumors.Hypermethylation of DNA can be demethylated.Therefore,it is possible that we may reverse the methylation of DAPK gene to treat tumors.Study on DAPK promoter methylation and its protein expression in cervical carcinoma may provide us a new way to understand the mechanisms of cervical carcinoma and new methods for diagnoses and treatment.In this study,we analyzed the relations between DNMT1 expression in cervical carcinoma and clinicopathologic factors by detecting DNMT1 mRNA expression in cervical carcinoma and HeLa cells;we detected the methylation status of DAPK promoter methylation in these samples and correlated to DAPK protein expression;we observed the inhibition effect of DNMT1 gene in HeLa cells that have being transfected, as well as the effect on proliferation and apoptosis of HeLa cells using recombinant interference plasmid pshRNA-dnmt1 for transfection.Based on the above results,we aimed to know more about the mechanism of cervical carcinogenesis,to find the potential molecular target for the therapy of cervical carcinoma,and to provide a theoretical basis for gene therapy of cervical carcinoma.The experiment was divided into the following three parts.The first part:DNMT1 expression in cervical carcinoma and its clinicopathological valueMethods1.In situ hybridization was used to detect the expression of DNMT1 mRNA in all the tissue samples;the relations between DNMT1 expression and clinicopathologic factors in cervical carcinomas were analyzed.2.RT-PCR was applied to detect DNMT1 mRNA of HeLa cells.3.Western blot was performed to examine the protein expression of DNMT1 gene in HeLa cells.4.Statistical analysis:using SPSS 13.0 statistical software package to analyze the experimental data.Differences in frequency among groups were evaluated by the Chi-Square test,takingα=0.05 as significant inspection standards.Results1.Positive rates of DNMT1 mRNA expression in normal uterine cervical tissues, CINs and cervical carcinomas were 10.0%,63.3%and 78.8%,respectively,with a significant difference(χ~2=29.057,P=0.000);The positive rate of DNMT1 mRNA expression in CIN tissues was statistically higher than in normal cervical tissues (χ~2=17.067,P=0.000);There was no significant difference between the positive rate of DNMT1 mRNA expression in CIN and cervical carcinoma(χ~2=3.226,P=0.072).2.There were no significant correlations of DNMT1 mRNA expression with histological type,grade of differentiation,FIGO stage and lymph node metastasis(χ~2=0.601,P=0.438;χ~2=2.160,P=0.340;χ~2=1.387,P=0.239;χ~2=0.578, P=0.447,respectively).3.RT-PCR and Western blot all showed DNMT1 mRNA and protein expressions in HeLa cells.The second part:DAPK methylation and DNMT1 expression in cervical carcinomaMethods1.The methylation status of DAPK gene promoter in cervical carcinomas,CINs, normal cervical tissues and cervical cancer HeLa cells was detected by Methylation-specific polymerase chain reaction(MSP),and their correlations with clinocopathological factors were analyzed.2.DAPK protein expression was detected by immunohistochemistry in cervical carcinomas,CINs and normal cervical tissues,and the relationship between DAPK protein expression and clinicopathologic factors of cervical carcinoma was analyzed.3.The relationship between DAPK promoter methylation and protein expression was analyzed,as well as the relevance between DNMT1 expression and DAPK methylation.4.Statistical analysis:using SPSS 13.0 statistical software package to analyze the experimental data.The positive rate of count data usingχ~2 test,the relationship between the two variants using Spearman correlation analysis,takingα=0.05 as significant inspection standards.Results1.The positive rate of DAPK protein expression in cervical carcinoma(40.4%, 21/52) was statistically lower than in normal cervical tissues(100.0%,20/20),CIN tissues(75.0%,45/60).There were significant differences between the positive rate of DAPK protein expression in normal cervical and CIN tissues,as well as in CIN and cervical carcinoma(χ~2=4.622,P=0.032;χ~2=13.791,P=0.000,respectively).2.DAPK protein expression in tumor stagesⅢ-Ⅳ(14.3%,2/14) was lower than less advanced tumors in stagesⅠ-Ⅱ(50.0%,19/38),with significant difference(χ~2=4.038, P=0.044).There were no significant correlations of DAPK protein expression with histological type,grade of differentiation and lymph node metastasis(χ~2=1.231,P=0.267;χ~2=0.289,P=0.866;χ~2=1.838,P=0.175,respectively).3.DAPK promoter methylation rate in cervical carcinoma(65.4%,34/52) was higher than that in CINs(18.3%,11/60) and normal cervical tissues(0%,0/20),with statistical significance(χ~2=39.639,P=0.000).There was a significant difference between in cervical carcinoma and CIN tissues(χ~2=25.658,P=0.000),no significant difference between in CINs and normal cervical tissues(χ~2=2.846,P=0.092).4.DAPK promoter methylation rate was higher in cervical squmous carcinoma (80.0%,32/40) than in adenocarcinoma(16.7%,2/12),with statistically significance (χ~2=13.680,P=0o000).No significant correlations between DAPK gene hypermethylation and tumor stages,grade of differentiation,and lymph node metastasis (χ~2=1.010,P=0.604;χ~2=0.000,P=1.000;χ~2=0.047,P=0.828 respectively).5.The expression of DAPK gene was lost in 88.2%(30/34) cases of cervical carcinoma with DAPK promoter methylation,but only in 5.6%(1/18) cases without promoter methylation.The occurrence of DAPK promoter hypermethylation was correlated strongly with a marked decrease in protein expression(r=0.802,P=0.000).6.The expression of DNMT1 mRNA was significantly correlated with CpG islands methylation of DAPK promoter(r=0.308,P=0.000).7,Promoter methylation of DAPK gene was detected positively in HeLa cell line.The third part:The construction of pshRNA-DNMT1 and effect of biology behaviour on human cervical carcinoma strain HeLaMethods1.Three targeted DNA fragments were designed,and synthesized into complementary chains by annealing,respectively.The obtained products containing short hairpin structure were inserted into plasmid vector pSilencer3.1-H1 with H1 promoter.The recombinant plasmids were transformed into Escherichia coli strain JM 109 for amplifying,identification of recombinant plasmid carried out with enzyme digestry EcoRⅠ/HindⅢand sequencing.2.EndoFree plasmid kit was used to extract recombinant plasmid pshRNA-DNMT1-A,B and C.3.Recombinant plasmid pshRNA-DNMT1-A,B and C was respectively transfected into HeLa cells with Lipofectmaine 2000.RT-PCR and Western blot were adopted to select one recombinant plasmid which showed the most optimal inhibition effect.4.RT-PCR and Western blot were performed to examine the mRNA and protein expression ofDNMT1 gene in HeLa cells after transfection 24h,48h and 72h.5.DAPK promoter methylation of HeLa cells after transfection was detected with MSP.6.The porliefration of the HeLa cells transfected with recombinant plasmid pshRNA- DNMT1 was investigated with CCK-8 kit7.Cell cycle and apoptosis of HeLa cells after transfection were detected with Flowcytometry(FCM).8.Statistical analysis:using SPSS 13.0 statistical software package to analyze the experimental data.Measurement data using the standard deviation of the number of disability and comparing among more than a few samples using one-way ANOVA. Takingα=0.05 as significant inspection standards.Results1.The three DNMT1-targeted shRNAs were successfully inserted into the plasmid vector pshRNA,and the coding sequences of the obtained shRNAs were consistent with the designed fragments.2.The screening results indicated that both recombinant plasmid pshRNADNMT1-A and B could effectively knock down the expression of DNMT1 gene in human cervical cancer cells,of which pshRNA- DNMT1-B was the better choice.No effect of pshRNA-DNMT1-C was seen.3.The RT-PCR results showed that the expression of DNMT1 mRNA appeared to decrease(inhibition rate:24.39%) 24h after transfection with pshRNA-DNMT1-B, showed significant change(64.43%) after 48h and was dramatically depleted(80.63%) after 72h.Likewise,the analysis by Western blot indicated that pshRNA- DNMT1-B induced a 31.13%inhibition of DNMT1 protein after 24h treatment,72.89%after 48h, and 87.36%after 72h.Both the results of RT-PCR and Western blotting showed statistical significance.4.Promoter methylation and unmethylation of DAPK were detected positively in the HeLa cells transfected with the recombinant plasmid.Only the promoter methylation were detected positively in cells transfected with empty vector and non-transfected cells.5.CCK-8 results showed that the growth of the HeLa cells transfected with the recombinant plasmid was inhibited significantly.There were statistical difference of absorbance value in cells transfected with recombinant plasmid comparing with non-transfected cells at 24h,48h,72h,96h,120h,but no significant difference was found in cells transfected with empty vector comparing with non-transfected cells(P＞0.05).6.Flow cytometry results showed that G0/G1 phase cells increased and S phase cells decreased after transfection with recombinant plasmid at 24h,48h,72h comparing with non-transfected,with statistically significant difference(P＜0.05).No significant difference was found between empty vector transfected cells and non-transfected cells (P＞0.05).7.Statistically,apoptosis rate of cells transfected with recombinant plasmid at 24h, 48h,72h comparing with non-transfected were significant different(P＜0.05),and the apoptosis rate of cells transfected with recombinant plasmid compared between different time points were significant different,72h group was the highest(P＜0.05);the difference of the apoptosis rate between transfected with empty vector and non-transfected was not significant(P＞0.05).Conclusions1.Increased expressions of DNMT1 mRNA from normal uterine cervical tissues, CINs to cervical carcinomas,together with positive expression of DNMT1 mRNA and protein in HeLa cells confirmed by RT-PCR,immuocytochemistry and Western blot indicate that DNMT1 may play a role in the development and progression of cervical carcinoma.2.DAPK protein expression level is lower in cervical carcinomas than that in CINs and normal uterine cervices.DAPK protein expressions are correlated with clinical stages. The results suggest that DAPK protein may play a role in the development and progression of cervical carcinoma.3.DAPK methylation rate is correlated with cervical epithelial malignant level and negatively correlated with DAPK protein expression.In addition,the promoter of DAPK is methylated in HeLa cell line,indicating that promoter methylation can inactivate DAPK and may contribute to the development of cervical carcinoma.4.The expression of DNMT1 mRNA was significantly correlated with CpG islands methylation status of DAPK promoter region.It suggests that DNMT1 may be a main cause of DAPK aberrant hypermethylation. 5.DNMT1 can be successfully silenced and DAPK promoter unmethylation can be partly restored by RNAi in cervical carcinoma cells.6.Downregulation of DNMT1 can inhibit proliferation of cervical carcinoma cells, delay the cell cycle at G0/G1 phase,and induce cell apoptosis.